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Introduction

The complex process of embryogenesis involves tight regulation of gene expression, which is controlled by different genetic and epigenetic modifications (1,2). Post-translational modifications of histones, including acetylation, methylation, ubiquitination and phosphorylation, are important epigenetic modifications regulating gene expression (3).

Methylation of lysine residues of histone molecules is an important part of epigenetics and is involved in the control of gene expression (4). The methylation state of a histone around a gene determines whether it is transcriptionally active or not; histone methylation is strictly controlled by histone lysine methyltransferases and demethylases (KDMs) that are responsible for transfer and removal of a methyl group (5). Methylation of histone H3 lysine 9 (H3K9) is a distinctive feature of transcriptionally repressed genes in eukaryotic chromatin (4,6). H3K9, which can be mono-, di-and tri-methylated (me1, me2 and me3, respectively), is well characterized for its roles in embryonic development (7). H3K9me3 is present in heterochromatin compartments (4) while H3K9me2 is present in euchromatin regions of eukaryotic chromatin (1). The meth-ylation status of H3K9 is maintained by two groups of KDMs containing Jumonji-C (JmjC) domains (8). Demethylases of the Kdm4 family catalyze H3K9me2/3 demethylation, while demethylation of H3K9me1/2 is catalyzed by Kdm3a (9-11).

Lysine demethylase 3a (Kdm3a), also known as jumonji domain containing 1A, JMJD1A, KIAA0742 and testis-specific gene A, is a JmjC-domain containing demethylase (8). In addition to the JmjC domain, Kdm3a contains a zinc finger domain and LXXLL motif; these domains/motifs are specific for steroid hormone receptor interactions (11,12). Kdm3a catalyzes demethylation of H3K9me1/2 and serves a role in the activation of gene transcription (8). The catalytic activity of Kdm3a, similar to other JmjC domain-containing proteins, involves a hydroxylation reaction requiring ferrous ion and α-ketoglutarate as cofactors (11,13).

Kdm3a has been implicated in the regulation of gene expression during numerous biological functions, including spermatogenesis, metabolism, sex determination, androgen receptor-mediated transcription and cell differentiation (14-17). Aberrant expression of kdm3a is associated with several malignancies, including colorectal cancer, breast cancer, hepatocellular carcinoma and hypoxia (18-21).

Multiple previous studies indicated the involvement of H3K9me2 demethylation in neural development (22-26). In human embryonic stem cells, Kdm3c was reported to serve roles in the repression of neural differentiation and was necessary for the maintenance of miR-302 expression (22). miR-302 is an epigenetic regulator of pluripotency and neural differentiation (27). Furthermore, depletion of lysine demethylase 1a splice variant catalyzing demethylation of H3K9me1/2, increases the level of H3K9me2 at the target promoters associated with the impairment of neuronal differentiation (23). In addition, lysine demethylases 7a and 7b which demethylate H3K9me1/2 are also involved in the regulation of neural development (24,25).

Xenopus laevis is a model used to investigate the mechanisms underlying neural development and differentiation (28,29). The present study investigated the developmental functions of Kdm3a and its effects on craniofacial and neural development during Xenopus embryogenesis. Whole-mount in situ hybridization (WISH) analysis revealed the expression of kdm3a in the anterior regions of Xenopus embryos, including the retina, central nervous system (CNS) and branchial arches. Morpholino antisense oligonucleotide (MO)-mediated knockdown of kdm3a resulted in defective craniofacial development and neural deformities. Furthermore, the results of WISH using neural crest-specific probes indicated that Kdm3a regulated neural crest migration as kdm3a morphant embryos exhibited decreased migration of neural crest cells compared with control MO-injected embryos. In addition, kdm3a depletion altered the expression of neural markers and downregulated the expression of genes specific for mesoderm development, cell adhesion and metabolism. The results of the present study suggest that Kdm3a may serve important roles during Xenopus embryogen-esis by regulating the expression of neural-specific genes.

Materials and methods

Xenopus husbandry and in vitro fertilization

Xenopus laevis (age, ~3 years old; 20 females and 10 males) were obtained from the Korean Xenopus Resource Center for Research and housed with a 12-h light/dark cycle at 18°C in containers built specifically according to the requirements for maintenance of laboratory organisms by the Institutional Review Board of Ulsan National Institute of Science and Technology (ethical approval no. UNISTACUC-16-14). To induce ovulation, Xenopus females were injected with 1,000 IU/animal of human chorionic gonadotropin (Dae Sung Microbiological Labs., Co., Ltd., Seoul, Korea) into the dorsal lymph sac in the evening. The following day, the abdomens of female frogs were gently squeezed and eggs were placed in 60-mm petri dishes containing 1X Modified Barth's Saline (MBS; 88 mM NaCl, 5 mM Hepes, 2.5 mM NaHCO3, 1 mM KCl, 1 mM MgSO4, and 0.7 mM CaCl2, pH 7.8). After washing three times with 0.1X MBS, eggs were fertilized using a suspension solution of sperm obtained from isolated testes of sacrificed males. Following successful fertilization, the embryos were swirled in a 2% L-cysteine solution (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) to remove the jelly coat and washed five times with 0.5X MBS. Live and healthy embryos were transferred to 0.5X MBS containing 2% Ficoll® PM 400 (GE Healthcare Life Sciences, Little Chalfont, UK) while unfertilized eggs and non-viable embryos were removed by observation under a light microscope (magnification, ×1).

Plasmids, mRNAs, MOs and microinjections of Xenopus embryos

A full-length kdm3a cDNA clone (GeneBank ID: NM_001095502) was obtained from the American Type Culture Collection (Manassas, VA, USA). The amplification of Flag-tagged kdm3a was performed by polymerase chain reaction (PCR), as previously described (30). The amplified fragment was sub-cloned into a pCS107 vector (Laboratory of Protein Dynamics and Signaling, National Cancer Institute, Frederick, MD, USA). Tagged kdm3a was subsequently linearized with PvuII restriction endonucleases. SP6 mMes-sage mMachine kit (Ambion; Thermo Fisher Scientific, Inc., Waltham, MA, USA) was used to synthesize capped mRNAs for microinjections. The kdm3a MOs and control MOs were synthesized by Gene Tools, LLC (Philomath, OR, USA; 5′-GTT CTC TTG CTG AGT GAG CAC CAT A-3′; Control MO, 5′-CCT CTT ACC TCA GTT ACA ATT TAT A-3′). Both blastomeres of two-cell staged embryos were microinjected with MOs (30 ng/blastomere) and/or mRNAs (1 ng) and incubated until the required stages (16, 22 and 32). For rescue experiments, a MO-resistant mRNA (kdm3a*) with seven point mutations in wobble codons followed by an ATG start codon (5′-ATG GTA CTG ACG CAA CAG GAA AAT-3′) was synthesized. For tissue-specific expression of neural crest-specific markers, kdm3a MO was microinjected along with β-galactosidase (Laboratory of Protein Dynamics and Signaling, National Cancer Institute; 300 pg) into one of the two blastomeres of two-cell stage embryos.

Whole-mount in situ hybridization (WISH) and β-galactosidase staining

Xenopus embryos were collected at developmental stage 16, 22 and 32 and prefixed in MEMFA (4% parafor-maldehyde, 0.1 M MOPS pH 7.4, 1 mM MgSO4 and 2 mM EGTA) for 2 h at room temperature. WISH was performed on fixed embryos by hybridization with neural crest-specific probes, including Twist-family bHLH transcription factor 1 (twist), snail family zinc finger 2 (snail2), transcription factor AP-2α (tfap2a), early growth response 2 (egr2) forkhead box d3 (foxd3), myc proto-oncogene, bHLH transcription factor (c-myc), msh homeobox 1 (msx1) and paired box 3 (pax3), as previously described (31). To distinguish the MO-injected side of the embryos, β-galactosidase staining was performed, as previously described (31).

Quantitative (q)PCR

Total RNA extraction was performed by lysing the embryos in Isol-RNA lysis reagent (5 Prime GmbH, Hamburg, Germany) and cDNA was prepared using the first strand cDNA synthesis kit (Takara Bio, Inc., Otsu, Japan) at 65°C for 5 min followed by 42°C for 1 h and 95°C for 5 min. qPCR reaction was performed using specific primers (Table I) and SYBR Premix Ex Taq, according to the manufacturer's protocol (Takara Bio, Inc.). Thermocycler was adjusted at 95°C for 30 sec to ensure denaturation, annealing temperature was set at 55°C for 30 sec followed by extension at 72°C for 1 min (30 cycles). The analysis was performed using StepOnePlus™ Real-Time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.). The relative expression levels of target genes were analyzed using the 2-ΔΔCq method (32). All data are representative of at least three experiments. Primers were designed using Primer3 software (33) and ornithine decarboxylase was used as an internal control.

Table I Primers used for qPCR.

Table I

Primers used for qPCR.

Gene	Primer sequence (5′→3′)
	Forward	Reverse
odc	CAG CTA GCT GTG GTG TGG	CAA CAT GGA AAC TCA CAC C
kdm3a	TCC TAC ACC TGC CTG CTC T	CAA GCT GGG CTG CAT TGG A
ag1	AAG GCC TGA AGA CCC TGG A	TGC GAG AGT CAG GGA TGG A
hesx1	AGC TTT CAC TAG GAG CCA GA	AGG TCC AAG GCT CTA TCA
zic3	CAA CAG TGA GGA ACC TTC CA	GGG CTT TGT TAG TCT GTA GC
six3	AAC ACG AGT CCA TCC TGC G	CCT GCT GGG GTT GGG GTA G
otx2	GGA TGG ATT TGT TAC ATC CGT C	CAC TCT CCG AGC TCA CTT CCC
en2	ATG AGC AGA ATA ACA GGG AAG TGG A	CCT CGG GGA CAT TGA CTC GGT GGT G
n-cam	GCG GGT ACC TTC TAA TAG TCA C	GGC TTG GCT GTG GTT CTG AAG G
foxg1	AGC AGC GAC GAT GTT TCA	GGC CAT TGG ACG TGG AGA A

[i] odc, ornithine decarboxylase; kdm3a, lysine demethylase 3a; Ag1, anterior gradient 1; hesx1, HESX hmeobox1; zic3, ZIC family member 3; six3, SIX homeobox 3; otx2, orthodenticle homeobox 2; en2, engrailed homeobox 2; n-cam, neural cell adhesion molecule 1; foxg1, forkhead box G1.

Animal cap assay

Xenopus embryos were microinjected with kdm3a MO and/or 2 ng of dominant negative bone morphogenetic protein 4 receptor (dnbr) mRNA (Laboratory of Protein Dynamics and Signaling, National Cancer Institute) which induced neural tissue formation from the prospective ectoderm. The animal caps were excised using forceps following removal of the chorion membrane from the vegetal side of late blastula (stage 8.5-9 embryos). Excised animal caps were cultured at 18°C in 0.5X MBS containing 2% Ficoll PM 400 until developmental stage 16 of Xenopus embryogenesis (34).

Alcian blue staining

For alcian blue staining, Xenopus embryos were collected at the late tadpole stage (stage 43) and fixed in Bouin's solution for 2 h at room temperature. Following fixation, embryos were washed in 70% ethanol + 0.1% NH4OH and stained with 0.05% Alcian Blue 8GX (Sigma-Aldrich; Merck KGaA) in 5% acetic acid for >2 h at room temperature. The embryos were subsequently washed in 5% acetic acid for 2 h at room temperature and cleared in 100% methanol. Following clearing, the embryos were kept in benzyl benzoate and benzyl alcohol at a ratio of 2:1, respectively.

Western blot analysis

The kdm3a MO-injected embryos were treated with the lysis buffer (137 mM NaCl, 20 mM Tris-HCl pH 8.0, 1% Nonidet-P40 and 10% glycerol) and 1 mM phenyl-methylsulfonyl fluoride (Amresco, LLC, Solon, OH, USA), 5 mM sodium orthovanadate (Sigma-Aldrich; Merck KGaA) and 1X protease inhibitor mix (Roche Diagnostics, Basel, Switzerland), followed by heating of lysates at 95°C in loading buffer for 5 min. Bradford assay was used for protein determination; lysates (40 µg/lane) were analyzed using SDS-PAGE (12% gel) followed by transfer to nitrocellulose membranes. The membranes were blocked with skim milk (5%) for 1 h at room temperature and proteins were detected with anti-Flag tag (cat. no. G188; Applied Biological Materials, Inc., Richmond, BC, Canada) for 1 h at room temperature. Subsequently, the membranes were incubated with goat anti-mouse (cat. no. sc2005; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) and goat anti-rabbit (cat.no. sc2004; Santa Cruz Biotechnology, Inc.) horseradish peroxidase-conjugated antibodies by incubating for 1 h at room temperature. Primary and secondary antibodies were used at a dilution of 1:1,000. The immunoreactive bands were detected using an enhanced chemiluminescence kit (HyGLO™ kit; Denville Scientific, Inc., South Plainfield, NJ, USA). Histone H3 (cat. no. ab1791; Abcam, Cambridge, UK) antibody is used as a loading control.

RNA sequencing

Total RNA was harvested from each sample (3 embryos/sample) at tadpole stage (stage 32) and RNA sequencing library was constructed according to manufacturer's protocol (TruSeq RNA Sample Prep kit v.2; cat. nos. RS-122-2001 and RS-122-2002; Illumina, Inc., San Diego, CA, USA) using polyA enrichment. For estimation of mRNA abundance, the reads were mapped to the Xenopus laevis cDNA sequences from the genome project consortium (35) using BWA software (v.0.7.15) (36), and the significant differential expression of genes was estimated using edgeR software (v.3.3.1). Genes exhibiting >4-fold change and false discovery rate <0.01 were considered significantly differentially expressed. To determine biological processes in which the differentially expressed genes were enriched, Fisher's test provided by the PANTHER database (release 20171205) (37) was used with human orthologous genes based on the best-hit results using BLASTP search (38). RNA sequencing raw data are available at the National Center for Biotechnology Information Gene Expression Omnibus database (accession no. GSE117754).

Statistical analysis

ImageJ software (v.1.45; National Institutes of Health, Bethesda, MD, USA) was used to analyze the data from the WISH and PCR analyses, and the results are presented as the mean ± standard error of the mean. Results obtained from three independent experiments. The level of significance was calculated using an unpaired t-test or one-way analysis of variance followed by Tukey's post hoc test using GraphPad Prism software (v.7; GraphPad Software, Inc., La Jolla, CA, USA). P<0.05 was considered to indicate a statistically significant difference.

Results

kdm3a is expressed at the anterior regions during Xenopus embryogenesis

To study the function of Kdm3a during Xenopus embryonic development, the present study investigated the expression pattern of kdm3a in early Xenopus embryos. qPCR analysis indicated that kdm3a was expressed at early stages of embryogenesis, ranging from the egg cell to the tadpole stage of development (Fig. 1A). However, markedly increased level of kdm3a expression was observed at the neurula stage (stage 17; Fig. 1A). The spatial expression of kdm3a was analyzed using WISH, and kdm3a mRNA localized to the CNS during the neurula stage of embryogenesis (stage 16; Fig. 1B). Furthermore, kdm3a was expressed at the anterior region, including the eye and the CNS at stage 22 (early tailbud stage), and was observed in the retina, otic vesicle and branchial arches at stage 32 (late tailbud stage) of Xenopus embryogenesis (Fig. 1B). The results of qPCR and WISH analysis indicate that kdm3a may serve roles during Xenopus embryogenesis and regulate neurogenesis during embryonic development.

Figure 1 Expression patterns of kdm3a during Xenopus embryogenesis. (A) Expression pattern confirmed by quantitative polymerase chain reaction. Ornithine decarboxylase served as the loading control. (B) Spatial expression pattern of kdm3a was analyzed by whole-mount in situ hybridization. Xenopus embryos were collected at stage 16 (neurula stage), 22 and 32 (tail bud stage) and analyzed under a light microscope (magnification, ×2.5). st., stage; kdm3a, lysine demethylase 3a.

Kdm3a knockdown arrests craniofacial formation during Xenopus embryogenesis

To investigate the physiological functions of Kdm3a during Xenopus embryogenesis, loss-of-function experiments were performed using kdm3a MO to inhibit kdm3a translation. Embryos at the two-cell stage were injected with kdm3a MO to repress kdm3a expression.

Embryos injected with the kdm3a MO exhibited malformed phenotypes, including defective head formation and markedly smaller eyes compared with the control MO-injected embryos (Fig. 2A). Anterior defects were identified in >90% of kdm3a morphants (Fig. 2B). Furthermore, abnormal pigmentation was observed among kdm3a morphants (Fig. 2C). Alcian blue staining was performed to visualize the effects of kdm3a MOs on craniofacial development. kdm3a morphants exhibited markedly smaller cartilage compared with cartilage formation in control embryos (Fig. 2C).

Figure 2 Depletion of kdm3a results in defective head formation and reduces cartilage formation. (A) kdm3a MOs were microinjected into two-cell stage embryos. Embryos were collected at stage 32 (late tailbud). (B) Quantitative analysis of embryos exhibiting defective head formation. (C) Alcian blue staining to visualize cartilage formation. Embryos were fixed at stage 43 (late tailbud). (D) Rescue experiments investigating malformed phenotypes induced by kdm3a depletion using microinjection of kdm3a* together with kdm3a MO. (E) Western blot analysis for kdm3a. H3 was used as the loading control. Embryos were analyzed under a light microscope (magnification, x2.5).***P<0.001 as indicated. Kdm3a, lysine demethylase 3a; kdm3a*, MO-resistant mRNA; MO, morpholino antisense oligonucleotide; H3, histone H3.

To confirm whether the malformed phenotypes were specifically induced by the depletion of kdm3a, rescue experiments were performed by microinjecting kdm3a* into two-cell stage embryos. Injection of kdm3a* recovered the malformed phenotypes observed in kdm3a morphants (Fig. 2D). This validation experiment confirmed that the defects observed in kdm3a morphants were caused by kdm3a knockdown during Xenopus embryogenesis. Furthermore, western blot analysis was used to examine protein expression of kdm3a. No kdm3a expression was observed in the group treated with MO, confirming the efficiency of knockdown (Fig. 2E). In addition, protein expression in embryos injected with kdm3a* or kdm3a* + MO, verified that MO did not bind to kdm3a* (Fig. 2E). These results suggested that the malformed phenotypes including defective craniofacial formation were caused by the loss of kdm3a, validating the role of this gene during Xenopus embryonic development.

Depletion of kdm3a affects neural crest migration during Xenopus embryonic development

The cells of neural crest emanate from neural tube immediately after its closure, and subsequently, migrate to specific regions of the embryo where they differentiate to the peripheral nervous system, facial skeleton cells and pigment cells (39,40). As described above, kdm3a morphants exhibited defects in craniofacial formation, abnormal head development and dense pigmentation (Fig. 2C), and kdm3a expression was observed at the neural plate during the neural stage of development (Fig. 1B). The present study further investigated whether kdm3a may affect neural crest migration.

To examine the role of Kdm3a in neural crest migration, kdm3a MO and β-galactosidase mRNA were injected into one blastomere of two-cell stage embryos. The injected sides of the embryos were visualized by β-galactosidase assays and WISH analysis was performed using neural crest marker genes. twist, snail2, tfap2a, egr2 and foxd3 are widely used neural crest marker genes (41,42). WISH expression patterns of twist (stage 32; late tailbud), and snail2, foxd3 and tfap2a (all at stage 20; neurula) indicated that injection with kdm3a MO significantly reduced neural crest migration on the injected side of the embryos (Fig. 3). In addition, kdm3a MO-injected sides of the embryos exhibited abnormal expression patterns of egr2-a, a hindbrain-specific marker; however, normal development of rhombomere 3 and 5, and neural crest was observed in the uninjected sides of the embryos (Fig. 3). These genes serve important roles during development of progenitor cells of neural crest into bona fide neural crest cells (43). Furthermore, twist is involved in neural crest cell migration as a repressor of transcription (44). Injected embryos exhibited abnormal expression patterns of twist, snail2 and foxd3, compared with the uninjected control (Fig. 3B).

Figure 3 Kdm3a is required for neural crest migration by regulating the expression of neural crest specifiers. (A) kdm3a MO and β-galactosidase mRNA were co-injected into one blastomere of two-cell stage embryos. Embryos were harvested at stage 16, 20 (neurula), and stage 32 (late tailbud). kdm3a depletion led to defective neural crest migration as indicated by the expression patterns of twist (stage 32; late tailbud), tfap2a, egr2, snail2, foxd3 (stage 20; neurula stage), c-myc, msx1 and pax3 (stage 16; neurula stage). Injected sides of the embryos exhibited reduced neural crest cell migration compared with the uninjected sides of the embryos. The black dot (•) represents the injected side. Embryos were analyzed under a light microscope (magnification, x2.5). (B) Expression patterns of neural crest specifiers, including twist, tfap2a, egr2, snail2, foxd3, c-myc, msx1 and pax3 induced by kdm3a knockdown. Data are presented as the mean ± standard error of the mean. ***P<0.001, as indicated. n.s, not significant; twist, twist-family bHLH transcription factor 1; snail2, snail family zinc finger 2; tfap2a, transcription factor AP-2α; egr2, early growth response 2; foxd3, forkhead box d3; c-myc, myc proto-oncogene bHLH transcription factor; msx1, msh homeobox 1; pax3, paired box 3; kdm3a, lysine demethylase 3a; MO, morpholino antisense oligonucleotide.

It has been previously demonstrated that Kdm3a may serve important roles in primary neuron formation; however, it was not required for neural induction (45). To verify these previous results, the present study observed the expression patterns of early markers of neural crest progenitor cells, including c-myc (46), msx1 and pax3 (47). Expression of c-myc was not affected by kdm3a knockdown (Fig. 3). Expression patterns of msx1 and pax3-neural crest induction markers (47,48) remained unaltered on the kdm3a MO-injected side (Fig. 3). Therefore, WISH analysis results together with the β-galactosidase assay demonstrated that depletion of kdm3a may affect the migration of neural crest cells by disturbing the expression of neural crest specifiers. Since facial cartilage is formed by the cranial neural crest and trunk neural crest cells differentiate into pigment cells (49,50); therefore, it may be hypothesized that the reduced size of cartilage and abnormal pigmentation in kdm3a morphants resulted from abnormal neural crest migration.

kdm3a knockdown alters the expression of neural-specific genes

Kdm3a serves essential roles in primary neuron formation and its depletion downregulates the expression of neural-differentiation associated genes (45). Therefore, the present study aimed to investigate the effect of Kdm3a on neural-specific (anterior, posterior and pan) genes. For this purpose, kdm3a MO and dnbr mRNA were co-injected into the animal pole region of two-cell stage embryos. Animal caps were excised from the microinjected embryos at blastula stage (stage 8.5-9) and cultured until neurula stage (stage 16). Xenopus animal caps, equivalent to embryonic stem cells, develop into the ectoderm, however, these cells are pluripotent and can differentiate into neural, mesodermal and endo-dermal tissues depending on the level and type of specific inducers (34,51). dnbr induces the formation of neural tissue from animal cap explants by inhibiting bone morphogenetic protein (BMP) signaling during early development of Xenopus and increasing expression of neurogenesis-specific genes (34).

Gene expression levels in animal caps dissected from kdm3a MO and dnbr mRNA co-injected embryos were analyzed by qPCR to determine the neural marker gene expression. Depletion of kdm3a led to the inhibition of expression of cement gland marker, anterior gradient 1 (ag1) (52), anterior markers, orthodenticle homeobox 2 (otx2), HESX hmeobox1 (hesx1) and forkhead box G1 (foxg1) (53), pan-neural marker, neural cell adhesion molecule 1 (n-cam) (48), and retina marker, SIX homeobox 3 (six3) (54), consistent with the kdm3a morphant phenotype (Fig. 4). By contrast, the expression levels of neural crest marker, ZIC family member 3 and posterior neural marker, engrailed homeobox 2 (55), were not suppressed by the knockdown of kdm3a (Fig. 4). Our qPCR data for neural marker gene expression are consistent with the previously published data (45). Furthermore, the present study indicated that kdm3a depletion inhibited the expression of six3. It may be hypothesized that kdm3a serves a role in the expression of retina specific markers at the early stage of embryonic development.

Figure 4 Kdm3a affects the expression of neural specific markers. Embryos were co-injected with kdm3a MO and dnbr mRNA into the animal pole region of two-cell stage embryos. Animal caps were dissected from injected embryos at stage 8.5-9 (blastula) and incubated in 0.5X MBS. Animal caps were collected at stage 16 (neurula) and processed for reverse transcription-quantitative polymerase chain reaction using standard methods to detect the expression levels of hesx1, ag1, foxg1, otx2, n-cam, zic3, six3 and en2. odc was used as the loading control. *P<0.05, **P<0.01 and ***P<0.001, as indicated. n.s, not significant; kdm3a, lysine demethylase 3a; MO, morpholino antisense oligonucleotide; dnbr, dominant negative bone morphogenetic protein 4 receptor; WE, whole embryos; ag1, anterior gradient 1; odc, ornithine decarboxylase; hesx1, HESX hmeobox1; zic3, ZIC family member 3; six3, SIX homeobox 3; otx2, orthodenticle homeobox 2; en2, engrailed homeobox 2; n-cam, neural cell adhesion molecule 1; foxg1, forkhead box G1.

The present study further conducted a transcriptomics analysis to elucidate the role of Kdm3a during Xenopus embryogenesis. Transcriptomics analysis demonstrated that kdm3a is important for metabolism, cell-cell adhesion and mesoderm development during Xenopus embryogen-esis (Fig. 5). Knockdown of kdm3a led to downregulation of a number of genes involved in mesoderm patterning and development, including fibroblast growth factor 13 (fgf13), signal transducer and activator of transcription 3 gene 1 (stat3) and T-box 5 (tbx5) (56). Furthermore, the depletion of kdm3a induced the downregulation of multiple genes involved in mesoderm induction and patterning (Fig. 5). Knockdown of kdm3a suppressed the expression of genes involved in cell-cell adhesion, including angiopoietin 2 (angpt2), cadherin 5 (cdh5) and fibrinogen β chain (fgb) (Fig. 5).

Figure 5 Metabolism, cell adhesion and mesoderm development. GO terms are affected by kdm3a knockdown. RNA-sequencing was performed and the enriched biological processes were analyzed for significantly downregulated genes (>4-fold change; FDR <0.01) following kdm3a knockdown. GO, Gene Ontology; kdm3a, lysine demethylase 3a.

qPCR analysis using animal caps and the transcriptomics data indicated that kdm3a depletion altered the expression of various genes involved in fundamental developmental processes. Therefore, Kdm3a may serve an important role in normal embryonic development of Xenopus laevis.

Discussion

Epigenetic regulator Kdm3a catalyzes demethylation of H3K9me1/2, and is involved in the maintenance of the histone code (15). It has been reported that this demethylase is involved in numerous biological and pathological processes, including germ cell development, sex determination, metabolism, stem cell differentiation, stem cell self-renewal and cancer epigenetics (2,57). In the present study, the expression pattern and physiological functions of Kdm3a were examined during embryonic development of Xenopus.

Appropriately regulated gene expression is necessary in the process of embryonic growth and development. Maternal and zygotic kdm3a was expressed during early stages of Xenopus embryonic development, until the tadpole stage of embryogen-esis. The kdm3a expression pattern observed in the present study is consistent with a previously conducted study (35). The qPCR analysis demonstrated that expression levels of kdm3a varied across developmental stages and were highest during the neurula stage (stage 17) of embryogenesis. kdm3a was expressed in the CNS during the neurula stage of embryonic development. The expression of kdm3a was observed in the anterior region, including the eye and the CNS at stage 22 (early tailbud), retina, otic vesicle, and branchial arches at stage 32 (late tailbud) of Xenopus embryogenesis. Microinjection of kdm3a MO into the two-cell stage Xenopus embryos induced phenotypic abnormalities, including reduced head size and smaller eyes, compared with control MO-injected embryos. Craniofacial development of kdm3a morphants was also affected, exhibiting markedly reduced cartilage and abnormal pigmentation, compared with the control embryos. Rescue experiments confirmed the role of Kdm3a during Xenopus embryogenesis. All malformed phenotypes of kdm3a morphants were rescued following co-injection of kdm3a MO and kdm3a*. These results indicate that Kdm3a may serve important roles during neural crest migration of Xenopus embryos.

Jmjc domain-containing demethylases are involved in neural development. Kdm7 is necessary for brain formation through its interaction with a BMP antagonist, the follistatin gene locus (26). Furthermore, Kdm6b is specific for demethylation of H3K27me3 and is involved in neural development (58). Based on the previous results regarding Kdm3a and its expression at the neurula stage (45), the present study investigated the physiological effect of Kdm3a during neural development. Injection of kdm3a MO altered the expression of neural crest specifiers. The WISH analysis indicated markedly altered expression of neural crest specifiers as a result of kdm3a knockdown. These results indicated that kdm3a depletion may be associated with impaired neural crest migration and may be required for facial cartilage formation.

Kdm3a serves a role in primary neuron formation and is known to regulate the expression of neuronal differentiation-associated genes; however the depletion of kdm3a exhibits no effect on the expression of neural stem progenitor markers pax6 and sox3 (45). The results of the present study supported these previously published data and indicated that Kdm3a may not be involved in the induction of neural crest cells, as demonstrated by the expression levels of early markers of neural progenitor cells, including c-myc, msx1 and pax3.

The present study analyzed the effect of Kdm3a on neural marker gene expression in animal caps of dnbr-injected embryos. Loss-of-function of kdm3a resulted in reduced expression of anterior neural markers hesx1, otx2 and foxg1, and pan-neural marker n-cam, suggesting that kdm3a may serve a role in the neural development during Xenopus embryogenesis. Transcriptomics data analysis of kdm3a morphants indicated that kdm3a may be required for mesoderm formation. The mesoderm is one of the earliest germinal layers and is essential for normal development of the skeletal system, muscular system and a major part of the neural system (59). Knockdown of kdm3a downregulated the expression of fgf13, stat3 and tbx5, and other genes associated with mesoderm development. The expression levels of angpt2, cdh5, fgb and other genes involved in cell-cell adhesion were also suppressed in kdm3a morphants.

In conclusion, the present study indicated that Kdm3a, an epigenetic modifier associated with histone code maintenance, is physiologically relevant as a regulator of neurogenesis during Xenopus embryonic development. Additional investigation is required to determine the interaction between this epigenetic regulator and developmental pathways, which may provide novel methods for the treatment of developmental disorders.

Funding

This study was supported by grants from the National Research Foundation of Korea (grant no. NRF-2015R1A2A1A10053265) and the Ministry of Science, ICT and Future Planning, the Republic of Korea (grant no. 2015R1A4A1042271).

Availability of data and materials

The datasets used and/or analyzed during the present study are available from the corresponding author on reasonable request. RNA sequencing raw data are available at the National Center for Biotechnology Information Gene Expression Omnibus database (accession no. GSE117754).

Authors' contributions

HKL, TI, CK and YK performed the experiments. HKL, TI, CK, JWP, OSK, BSK, DSL and HSL performed the data analysis and wrote the manuscript. TK, TJP and HSL designed the study, interpreted the results and critically analyzed the manuscript.

Ethics approval and consent to participate

Experiments were conducted according to the guidelines of the Animal Care and Use Committee consistent with international laws and policies (National Institute of Health Guide for the Care and Use of Laboratory Animals; NIH publication no. 85-23, 1985). The Institutional Review Board of Ulsan National Institute of Science and Technology in Korea approved the experimental use of amphibians (approval no. UNISTACUC-16-14). All members of the research group were trained for the appropriate care and use of experimental organisms. There were no unexpected cases of mortality of adult Xenopus during the present study.

Patients consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Acknowledgments

Not applicable.

References